Humoral immune response of flounder to Edwardsiella tarda: The presence of various sizes of immunoglobulins in flounder

Abstract

Antisera obtained from flounder Paralichthys olivaceus immunized with Edwardsleila tarda were fractionated using Sephacryl S-300 gel filtration. The flounder was found to contain various sizes of immunoglobulins (Igs) ranging from <200 to >700 kDa. These Igs were combined into 3 pools according to molecular weight; pool I consisted of high molecular weight (HMW) Igs of >700 kDa, pools II and III consisted of low molecular weight (LMW) Igs of 230 to 700 kDa (pool II) and <230 kDa (pool III). After being further purified by hydroxyapatite column chromatography and isoelectric focusing, HMW Ig in pool I and LMW Ig in pool III were compared with each other by SDS-PAGE under reducing conditions. They were found to be composed of 1 size of heavy chain and 2 sizes of light chain. While HMW Ig in pool I contained 68 kDa H chains and 22 kDa and 24 kDa L chains, LMW Ig in pool III contained 69 kDa H chains and 22 kDa and 26 kDa L chains. A set of 5 monoclonal antibodies (MAbs) against HMW Igs was prepared. The reactivities of these MAbs with Igs in the 3 pools were determined by enzyme linked immunosorbent assay (ELISA) and dot immunoassay. All 5 MAbs reacted strongly only to the HMW Igs. Results suggest that flounder lgs comprise LMW lgs as well as HMW Igs and the physicochemical and antigenical characteristics of LMW Igs are different from those of HMW Igs.